Label-free nonlinear optical signatures of extracellular vesicles in liquid and tissue biopsies of human breast cancer.

Extracellular vesicles (EVs) have been implicated in metastasis and proposed as cancer biomarkers. However, heterogeneity and small size makes assessments of EVs challenging. Often, EVs are isolated from biofluids, losing spatial and temporal context and thus lacking the ability to access EVs in situ in their native microenvironment. This work examines the capabilities of label-free nonlinear optical microscopy to extract biochemical optical metrics of EVs in ex vivo tissue and EVs isolated from biofluids in cases of human breast cancer, comparing these metrics within and between EV sources. Before surgery, fresh urine and blood serum samples were obtained from human participants scheduled for breast tumor surgery (24 malignant, 6 benign) or healthy participants scheduled for breast reduction surgery (4 control). EVs were directly imaged both in intact ex vivo tissue that was removed during surgery and in samples isolated from biofluids by differential ultracentrifugation. Isolated EVs and freshly excised ex vivo breast tissue samples were imaged with custom nonlinear optical microscopes to extract single-EV optical metabolic signatures of NAD(P)H and FAD autofluorescence. Optical metrics were significantly altered in cases of malignant breast cancer in biofluid-derived EVs and intact tissue EVs compared to control samples. Specifically, urinary isolated EVs showed elevated NAD(P)H fluorescence lifetime in cases of malignant cancer, serum-derived isolated EVs showed decreased optical redox ratio in stage II cancer, but not earlier stages, and ex vivo breast tissue showed an elevated number of EVs in cases of malignant cancer. Results further indicated significant differences in the measured optical metabolic signature based on EV source (urine, serum and tissue) within individuals.

Differentiation of breast tissue types for surgical margin assessment using machine learning and polarization-sensitive optical coherence tomography.

We report an automated differentiation model for classifying malignant tumor, fibro-adipose, and stroma in human breast tissues based on polarization-sensitive optical coherence tomography (PS-OCT). A total of 720 PS-OCT images from 72 sites of 41 patients with H&E histology-confirmed diagnoses as the gold standard were employed in this study. The differentiation model is trained by the features extracted from both one standard OCT-based metric (i.e., intensity) and four PS-OCT-based metrics (i.e., phase difference between two channels (PD), phase retardation (PR), local phase retardation (LPR), and degree of polarization uniformity (DOPU)). Further optimized by forward searching and validated by leave-one-site-out-cross-validation (LOSOCV) method, the best feature subset was acquired with the highest overall accuracy of 93.5% for the model. Furthermore, to show the superiority of our differentiation model based on PS-OCT images over standard OCT images, the best model trained by intensity-only features (usually obtained by standard OCT systems) was also obtained with an overall accuracy of 82.9%, demonstrating the significance of the polarization information in breast tissue differentiation. The high performance of our differentiation model suggests the potential of using PS-OCT for intraoperative human breast tissue differentiation during the surgical resection of breast cancer.

Flat epithelial atypia with and without atypical ductal hyperplasia: to re-excise or not. Results of a 5-year prospective study.

Flat epithelial atypia (FEA) of the breast have a tendency to calcify and, as such, are becoming increasingly detected by mammography. There is no consensus yet on whether to excise these lesions or not after diagnosis on core needle biopsies (CNB). We reviewed 3,948 cases of breast CNB between June 2004 and June 2009 correlating histomorphologic, radiological, and clinical features. There were 3.7 % (145/3,948) pure FEA and 1.5 % (58/3,948) concomitant FEA and atypical ductal hyperplasia (ADH). In the pure FEA population, 46.2 % (67/145) had microcalcifications on mammography with 65.5 % (95/145) of patients undergoing subsequent excisional biopsies with the following findings: benign 20 % (19/95), ADH 37.9 % (36/95), ductal carcinoma in situ (DCIS) 1.1 % (1/95), and DCIS and invasive ductal carcinoma (IDC) 2.1 % (2/95). In the concomitant FEA and ADH group, 86.2 % (50/58) patients had microcalcifications on radiograph with 74.1 % (43/58) of patients undergoing subsequent excisions with: benign 23.3 % (10/43), DCIS 9.3 % (4/43), DCIS and IDC 4.7 % (2/43), DCIS + lobular carcinoma in situ + invasive lobular carcinoma 2.3 % (1/43), and tubular carcinoma 2.3 % (1/43). The incidence of carcinoma in the FEA + ADH group is 18.6 % (8/43) and 3.2 % (3/95) for the pure FEA group. This difference is statistically significant (p = 0.0016). The relative risk of carcinoma in the ADH + FEA group versus the pure FEA group is 6.4773, with 95 % CI of 1.8432 and 22.76 24. Five-year mean follow-up in the unexcised pure FEA did not show any malignancies. These findings suggest that pure FEA has a very low association with carcinoma, and these patients may benefit from close clinical and mammographic follow-up while the combined pure FEA and ADH cases may be re-excised.